The Impact of Compressed Femtosecond Laser Pulse Durations on Neuronal Tissue Used for Two-Photon Excitation Through an Endoscope

Accurate intraoperative tumour margin assessment is a major challenge in neurooncology, where sparse tumours beyond the bulk tumour are left undetected under conventional resection. Non-linear optical imaging can diagnose tissue at the sub-micron level and provide functional label-free histopathology in vivo. For this reason, a non-linear endomicroscope is being developed to characterize brain tissue intraoperatively based on multiple endogenous optical contrasts such as spectrally- and temporally-resolved fluorescence. To produce highly sensitive optical signatures that are specific to a given tissue type, short femtosecond pulsed lasers are required for efficient two-photon excitation. Yet, the potential of causing bio-damage has not been studied on neuronal tissue. Therefore, as a prerequisite to clinically testing the non-linear endomicroscope in vivo, the effect of short laser pulse durations (40–340 fs) on ex vivo brain tissue was investigated by monitoring the intensity, the spectral, and the lifetime properties of endogenous fluorophores under 800 and 890 nm two-photon excitation using a bi-modal non-linear endoscope. These properties were also validated by imaging samples on a benchtop multiphoton microscope. Our results show that under a constant mean laser power, excitation pulses as short as 40 fs do not negatively alter the biochemical/ biophysical properties of tissue even for prolonged irradiation.